Detection and molecular characterization of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum from lambs and goat kids with hemorrhagic abomasitis in Turkey

Background The pathogenic Clostridia cause neurotoxic, histotoxic and enterotoxic infections in humans and animals. Several Clostridium species have been associated with abomasitis in ruminants. The present study aimed to investigate the frequency, and the presence of virulence genes, of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum in lambs and goat kids with hemorrhagic abomasitis. Results A total of 38 abomasum samples, collected from lambs and goat kids of 1 week to 1 month of age in different farms located in eastern Turkey between 2021 and 2022, were evaluated by histopathology, culture and PCR. At necropsy, the abomasum of the animals was excessively filled with caseinized content and gas, and the abomasum mucosa was hemorrhagic in varying degrees. In histopathological evaluation, acute necrotizing hemorrhagic inflammation was noted in abomasum samples. The examination of swab samples by culture and PCR revealed that C. perfringens type A was the most frequently detected species (86.84%) either alone or in combination with other Clostridium species. P. sordellii, C. perfringens type F and C. septicum were also harboured in the samples, albeit at low rates. Beta2 toxin gene (cpb2) was found in three of C. perfringens type A positive samples. Conclusion It was suggested that vaccination of pregnant animals with toxoid vaccines would be beneficial in terms of protecting newborn animals against Clostridial infections. This study investigated the presence of clostridial toxin genes in abomasal samples for the first time in Turkey.

Epidemiological investigations may help the development of vaccination programs against clostridial diseases and a better understanding of pathogenicity of Clostridium species. Although many studies have been conducted on the role of Clostridium species in the etiology of abomasitis in calves, there is a paucity of information in terms of etiology of abomasitis in lambs and goat kids. P. sordellii has been isolated from abomasum lesions of lambs in Turkey (11). The isolation of C. perfringens type A from lambs with enteric diseases has also been reported in Turkey and Iran (32,35). However, to our knowledge, no studies are available concerning the presence of Clostridial toxin genes in abomasal samples in Turkey. This sudy was carried out to investigate the presence of C. perfringens, Clostridium septicum and P. sordellii and their toxin genes in abomasal samples of lambs and goat kids with hemorrhagic abomasitis in Turkey.

Necropsy and histopathological findings
Necropsies revealed that gross findings were almost similar in all animals. The most striking finding at necropsy was that the abomasums of the animals were excessively dilated with caseinized content and gas, and multifocal black foci were seen in serosal surface ( Fig. 1 A). When the abomasum mucosa was opened, it was observed that these black foci were petechial hemorrhages in varying degrees (Fig. 1B). The hemorrhages were quite severe in some animals and were observed to cover the entire abomasum mucosa like a layer, and the abomasal wall was thickened duo to edema ( Fig. 1 C). Many erosions and ulcers were observed on the abomasal mucosa after the contents were removed (Fig. 1D). Ecchymotic hemorrhages were also observed in the epicardium and endocardium of the heart in addition to hemorrhages in the abomasum of five lambs and three kids.
Acute necrotizing hemorrhagic inflammation of abomasum mucosa was observed at microscopic examination. Moderate or severe hyperemie, congestion, edema and hemorrhagies were found in the propria and submucosa (Fig. 2 A). There were erosions (Fig. 2B), some of which extending into submucosa ulcers characterized with epithelial degenerasion/necrosis and desquamation. Mild inflammatory infiltration including neutrophils, lymphocytes and macrophages was seen in the propria mucasa of the abomasums (Fig. 2 C). In addition, many large rod-shaped bacteria were detected in the debris and mucosal surface of abomasum in seven lambs and four kids (Fig. 2D). These bacteria were gram positive in the staining made by gram staining method.

Molecular findings
Out of 38 samples, 33 (86.84%) were found positive for C. perfringens type A alone or in combination with other Clostridium species. C. perfringens type F (cpa and cpe gene positive) was detected in two (5.26%) samples.
Other types of C. perfringes were not detected in any of the samples. Three samples were positive for cpb2 in addition to cpa. C. perfringens type A and P. sordellii were detected in five (13.15%) samples. Two (5.26%) samples were positive for only P. sordellii and one (2.63%) sample for C. perfringens type A, P. sordellii and C. septicum ( Table 1). The genes tcsL and tcsH were not detected in P. sordellii positive samples. One (2.63%) of the samples was found to be negative for either of these bacteria.

Discussion
Clostridial abomasitis is characterized by necrosis of abomasal mucosa caused by exotoxins produced by several species of Clostridium genus in the gastrointestinal tract of animals [1]. Overgrowth of Clostridium species within the gastrointestinal tract and subsequent exotoxin release may lead to sudden death in animals under predisposing conditions [2,29]. Factors such as overfeeding, contamination of pooled colostrum, poor hygiene in farms, changes in diet, mineral deficiencies and formation of trichophytobezoars predispose to abomasal bloat and abomasitis in young ruminants [2]. Although C. septicum is traditionally associated with abomasitis in ruminants, the role of other clostridial agents is not well understood. The present study aimed to investigate the frequency, and the presence of virulence genes, of C. perfringens, P. sordellii and C. septicum in lambs and goat kids with hemorrhagic abomasitis. The vast majority of the 38 abomasal samples were determined to harbour C. perfringens type A. In addition, P. sordellii, C. perfringens type F and C. septicum were detected in the samples, albeit at low rates. Meanwhile, a few samples contained more than one Clostridium species. These findings put forward that C. perfringens type A has an important role in the etiology of hemorrhagic abomasitis in small ruminants. Recently, C. perfringens type A was increasingly isolated from calves with abomasitis [6][7][8]. In the USA, C. perfringens was reported to be present in approximately 67% of calves died of emphysematous abomasitis and abomasal bloat [16]. On the other hand, the number of studies reporting the presence of C. perfringens in abomasitis cases of lambs and goat kids was rather limited. In accordance with our results, C. perfringens type A was detected in 84% of the cases with clostridial enterotoxaemia in lambs and goat kids in Italy [15]. In another study carried out in Pakistan, the majority of the C. perfringens isolates from sheep and goats (healthy and diseased) were characterized as type A (82%) [30]. Likewise, 81% of C. perfringens isolates obtained from domestic livestock were reported to belong to type A in Saudi Arabia [31]. In Turkey, 77% of C. perfringens isolates from lambs suspected of enterotoxemia were genotyped as type A [32]. C. perfringens types B, C and D toxoid vaccines produced in Turkey are used against C. perfringens infections in animals. However, the results of the current study Fig. 1 Macroscopic view of abomasums. A: Excessively distended abomasum with gas and caseinized contents, and multifocal hemorrhagic foci seen from serosa in a one-week old lamb. B: Multifocal severe petechyial hemorrhages on mucosal surface of the abomasum. C: The entire abomasum mucosa was covered with hemorrhagic content and abomasal wall were thickened duo to edema in a two-week old goat kid. D: Many erosions and ulcers were observed on the abomasal mucosa after the contents were removed in a ten-day old lamb suggest that C. perfringens type A toxoid vaccine should also be considered in the vaccination programs of sheep and goats.
The major toxin of C. perfringens type A is alpha toxin which is a zinc metallophospholipase. High concentrations of this toxin can damage plasma membrans of host cells [2]. It was reported that alpha toxin might contribute to pathogenesis of bovine necrohemorrhagic enteritis [33]. However, the role of C. perfringens type A in the pathogenesis of enteric disease of animals is still conroversial, as it can be found in the intestine of clinically healthy animals [34]. In recent years, beta2 toxin has frequently been detected in C. perfringens isolates from animals and humans [14,15,35]. Also, a significant relationship has been reported between cpb2-positive C. perfringens isolates and diarrhoea in pigs [36]. This toxin has been detected in horses with intestinal disorders, as well [37]. In the present study, although only three of C. perfringens type A positive samples were found to harbour cpb2, it can be suggested that beta2 toxin might act in sinergy with alpha toxin in the pathogenicity of C. perfringens type A [1,14,33]. However, it should be underlined that some studies failed to detect cpb2 in C. perfringens type A isolates from either bovine clostridial abomasitis (BCA) or jejunal hemorhage syndrome (JHS) [7,31].  According to a recently introduced toxin-based typing system, C. perfringens strains containing cpa and cpe genes were classified as type F, while those containing cpa and netB genes were as type G [12]. C. perfringens type F is associated with food poisoning in humans. However, a few enterotoxigenic infections caused by type F strains have been reported in animals [38,39]. Although the presence of cpe has been reported in a goat with necrotizing enterocolitis [40], the role of enteroxin producing C. perfringens strains in animal diseases is not enlightened [39]. Our results suggested that enterotoxin does not have an important role in hemorrhagic abomasitis cases, but further studies are needed to clarify the pathogenicity of enterotoxin in animal diseases. C. perfringens type G causes necrotic enteritis in poultry [2]. The presence of this type has also been reported in a cow [41]. However, in accordance with many previous studies [7,14,42,43], none of the samples were positive for C. perfringens type G in the present study.
Although P. sordellii is known to cause gas gangrene in animals [2], it has also been isolated from lambs with abomasitis [10,11,44]. In the current study, P. sordellii was detected as the second most common bacterial species after C. perfringens type A. Lethal and hemorrhagic toxins (TcsL and TcsH) are considered to be the main virulence factors of P. sordellii. These toxins are members of the Large Clostridial Cytotoxin (LCC) family [45,46]. Although at low rates, the presence of LCC genes in P.sordellii strains isolated from clinical cases including horses with enterocolitis has been demonstrated in the UK, USA and Australia [23,46]. However, this study failed to show these genes in P. sordellii isolates. Similarly, Zerrouki et al. [47] reported that P. sordellii isolates originated from a hospital environment in Algeria did not harbour either TcsL or TcsH toxins. The absence of these genes in our isolates was not surprising due to the facts that the number of isolates was small (n = 8) and that the majority of P.sordellii strains do not encode LCC genes [46]. It is therefore possible that additional virulence factors can contribute to the pathogenicity of P. sordellii. However, it is known that nontoxigenic strains of P.sordelli can cause the disease, albeit at low severity [48,49].
Clostridium septicum is considered as the etiological agent of braxy in sheep [2]. It has been determined in lambs and calves with suppurative abomasitis [4,9]. Acute haemorrhagic abomasitis due to C. septicum has also been reported in experimental sheep [3]. However, the results of this study suggested that C. septicum was not the primary agent of abomasitis in lambs and goat kids in Turkey. It is known that small ruminants are mostly vaccinated against braxy in Turkey. Therefore, the detection of this agent in only one sample in the present study may be attributed to passive immunity in newborns. Similar results have also been reported elsewhere. For instance, C. septicum was not detected from lambs aged between 2 and 5 weeks with abomasal haemorrhage and ulcers in Norway [10].
Other bacterial agents such as Clostridium fallax have rarely been reported in abomasitis cases of lambs [10,20]. Recently, Sarcinia genus bacteria have also isolated from lambs and calves with abomasitis [10,19,20]. In the present study, the samples were not analyzed for either C. fallax or Sarcinia spp. However, the organisms consistent with Sarcinia spp. were not observed on the surface or within the abomasum wall in the histopathological examination.

Conclusion
The results of this study showed that C. perfringens type A was the most common species followed by P. sordellii in lambs and goat kids with hemorrhagic abomasitis in eastern Turkey. Interestingly, C. septicum was detected at the lowest rate in the abomasal samples, probably due to widespread vaccination of mothers. Also, the present study provided data concerning clostridial toxin genes in abomasum samples for the first time in Turkey. Further comprehensive studies are needed to have a better understanding of the pathogenic mechanisms of Clostridium species in abomasitis of small ruminants.

Sampling
The study material consisted of 21 lambs and 17 goat kids aged between 1 week and 1 month belonging to 30 different farms located in Eastern Turkey, which were submitted to the Pathology Department of Firat University between 2021 and 2022. Sudden onset of weakness, inability to suck the mother, reluctance to move or rise, swelling in the abdomen and death within 2-3 h to 1 day were recorded as general complaints in animals that were born healthy and received sufficient amount of colostrum. The mortality rate of the offsprings in the abovementioned age groups was informed to be around 15-20% in the flocks.

Necropsy and histopathological examination
Systematic necropsies were performed, tissue samples were taken from all animals, and fixed in 10% buffered neutral formalin solution. After routine procedures, the prepared paraffin blocks were cut into 3 μm thick, stained with haematoxylin and eosin (H&E), Brown and Brenn method for gram staining, and were evaluated by light microscopy.

Bacteriological culture
The swab samples were cultured onto 5% blood agar and incubated in an anaerobic jar for 48 h at 37 °C. Following Gram staining, suspected colonies were subcultured into cooked meat medium and incubated for 48 h at 37 °C in anaerobic conditions. Anaerobic media were supplied with anaerobic gas kits (Anaerocult A, Merck).

DNA extraction
DNA extraction was carried out from the bacterial culture in the cooked meat medium. For this, 300 µL of bacterial cultures were transferred to Eppendorf tubes. Each sample was treated with 300 µl TNES buffer (20mM Tris, 150 mM NaCl, 10 mM EDTA, 0.2% SDS) and 6 µl proteinase K (20 mg/ml), and then inactivated at 56 °C for 2 h. After the mixture was boiled for 10 min., 600 µL phenol-chloroform-isoamyl alcohol was added. The mixture was shaken vigorously for 5 min and centrifuged at 11600x g for 10 min. The upper phase was transferred to another Eppendorf tube and 3 M sodium acetate (0.1 volume) and ethanol (2.5 volume) were added. The mixture was vortexed and kept at -20 °C overnight. The mixture was then centrifuged at 11600x g for 10 min, and supernatant was removed. The pellet was washed with 70% ethanol and centrifuged at 11.600 g for 5 min. Finally, the pellet was dried for 45 min and suspended in 50 µL distilled water.

Polymerase chain reaction (PCR)
C. perfringens NCTC 8239 (cpa + and cpe + ), C. perfringens NCTC 13,110 (cpa + , cpb + cpb2 and etx + ), C. perfringens CCUG 44,727 (cpa + and itx + ), C. septicum genomic DNA and P. sordellii genomic DNA were used as positive controls. A multiplex PCR was performed for the detection of alpha (cpa), beta (cpb), epsilon (etx), iota (itx), enterotoxin (cpe) and beta2 (cpb2) toxin genes of C. perfringens in a thermal cycler (Techne, Staffordshire, UK), in a total reaction volume of 25 µL containing 12.5 µL 2X AmpMasterTaq master mix (AmpMasterTMGeneAll Biotechnology, Cambiol, Cambridge, Cat No: 541 − 010), 2.5 µL of DNA, and 1 µL of each specific primer (10 µM). The amplified products were electrophoresed in a 1.5% agarose gel containing 10 µL ethidium bromide solution. Amplified products were visualized and photographed under UV light. PCR products with the molecular sizes of approximately 402 bp, 196 bp, 567 bp, 655 bp, 293 and 506 bp were considered positive for cpa, cpb, cpb2, etx, itx and cpe genes, respectively. A single PCR was employed for the detection of the C. perfringens necrotic enteritis B-like toxin (netB) gene. A primer pair specific for the flagellin gene (fliC) was used for the identification of C. septicum by a single PCR. The C. septicum positive sample was also screened for the presence of alpha toxin (Hemolysin) gene. For the identification of P. sordellii, the samples were tested with PCR as previously described [50]. The primers of JRP4589 and JRP4590 were used to amplify a fragment of approximately 470 bp, representing an internal region of sdlO gene. PCR was also performed to determine the presence of the lethal toxin (tcsl) and hemorrhagic toxin (tcsh) genes of P. sordellii as previously described [29]. The primers used in this study were listed in Table 2.

HE
Hematoxylin-eosin PCR Polymerase chain reaction Table 2 DNA sequences of primers used in this study